Project Summary The DNA damage Response (DDR) is a regulatory network that coordinates cellular processed in response to DNA damage and replication stress. It also directly orchestrates DNA repair choices by sensing different DNA damage structures and transduces that information in cis to activate specific and appropriate repair options, thus optimizing repair. DDR's importance is highlighted by the many cancer predisposition syndromes resulting from its inactivation, including hereditary breast caner as well as its role in promoting sensitivity to genotoxic chemotherapy and synthetic lethality with the successful treatment of BRCA1/2-deficient tumors with PARP inhibitors. In the last 25 years we, and others, have investigated the composition of the central sensing and signaling apparati that detect and respond to genotoxic stress in yeast and now mammals. This revealed a conserved core of sensing and signaling proteins. Our analysis of substrates of this DDR kinase cascade has revealed an extremely diverse set of proteins and functions contacted by the DDR in mammals and the great majority of these activities are not conserved in yeast. This includes proteins like RHINO, Abraxas, SMARCAL1, RANZB3, WHSC1, NBA1/MERIT40, RAP80, the entire Fanconi anemia pathway including FANCI, FAN1, foci regulatory proteins Mdc1, p53BP1, RNF8, RNF168/RIDDLIN, RFWD3 not to mention other key DDR patheays like p53/ p21, USP28 and BDR7. Our analysis of ATM and ATR substrates and our and other's analyses of non-ATM/ATR regulated phosphorylation events have implicated over 1000 proteins in the DDR, the vast majority of which have no previous links to the DDR and have no yeast counterparts. Therefore, we think there are many, many new components of the DDR to be discovered in mammals and it is critical that we set out to identify these factors in order to generate a complete understanding of the DDR and its significance in cellular and organismal physiology. This includes a new effort in cellular senescence, a key response to DNA damage that prevents tumorigenesis and promotes aging. Toward this end, we have developed sophisticated genetic tools that allow us to employ RNAi, CRISPR and ORF expression to find new protein candidates involved in promoting survival in response to DNA damage. We have performed three preliminary screens for loss of function and gain of function to find new DDR candidate proteins that will serve as the basis for AIM1 and AIM2. In these AIMs we propose to carry out exhaustive validation of the candidates to identify bona fide new DDR proteins and will follow up on two candidate proteins already validated. In addition we have uncovered a key protein in regulation of the senescence secretory phenotype, GATA4, which responds to ATM and ATR to activate NFkB. We will further explore its role in senescence and a new protein required for senescence, ZNF292. These innovative methods will allow us to deeply probe the layers of the DDR including senescence to identify and prioritize new proteins and to then probe the functions of these proteins in the DDR using approaches we have pioneered for so many years. !!